Project Summary/Abstract Pancreatic cancer is the 4th leading cause of cancer deaths in the US with a 5-year survival rate of less than 7% and a median survival of only 3 to 6 months. The majority of pancreatic ductal adenocarcinomas (PDACs) are diagnosed at a late stage, are not surgically resectable, and respond poorly to chemotherapy, so an emphasis on earlier detection methods is critical. The secreted factor Sonic Hedgehog (SHH) has been identified as a protein that contributes to early tumor formation and is produced by tumor cells to promote the development of the dense stromal component of PDAC. Our goals are to identify SHH pathway components whose expression contributes to PDAC initiation and development, and define how SHH signaling promotes PDAC development in response to a high cholesterol diet. In preliminary work, we have identified the SHH binding protein CDON as an early marker of pancreatic lesions. Our recent work in Drosophila shows that Boi, the fly homolog of CDON, functions to control Hedgehog (Hh) levels via a novel sequestration and release mechanism. Boi acts as a rheostat, sequestering Hh when dietary cholesterol levels are low, and releasing Hh upon feeding, when cholesterol levels increase. Importantly, we find that cholesterol is sufficient to trigger SHH release from human PDAC cells, suggesting that this mechanism is conserved in cancer. We propose that cholesterol-stimulated SHH release promotes stromal activation in early pancreatic lesions, thus promoting the development of early non-cancerous lesions into adenocarcinoma. In addition, we hypothesize that CDON expression is elevated in early preneoplastic lesions, and that this suppresses the transition from benign to adenocarcinoma. We will test this by 1) defining the role of CDON in cholesterol-stimulated SHH sequestration and release in human PDAC cells, and 2) determining how CDON influences the development of adenocarcinoma from benign lesions in a KRAS-dependent mouse model. A combined approach of reducing cholesterol and treating patients with kinase inhibitors to target SHH signaling in tumor cells may maintain a benign state and prevent progression into cancer. This work will serve as the foundation for future studies aimed at developing translational approaches for combined diet and drug treatment for PDAC prevention.